Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

International Journal of Molecular Sciences

GaneshS. Anand¹  NikhilK. Tulsian¹  ValerieJia-En Sin²  Hwee-Ling Koh² 

[1] Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Singapore;Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore;

关键词: phosphodiesterase (PDE);    natural products;    inhibitors;    protein kinase;    selectivity;    fluorescence polarization;   

DOI  :  10.3390/ijms22105242

来源: DOAJ

【 摘 要 】

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–R_D complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

【 授权许可】

Unknown